Article Figures & Data
Figures
- Figure 1.
Construction and confirmation of the mcDNA vector containing the PSCA-CAR construct. A, Illustrative representation of the PSCA-CAR construct. B, A cartoon showing the process to generate mcDNA-PSCA-CAR. The PSCA-CAR construct was cloned into MN511A-1 to form the parental plasmid pMC-Easy-PSCA-CAR (from left to middle). After L-arabinose induction, pMC-Easy-PSCA-CAR was separated into mcDNA-PSCA-CAR and the bacterial backbone which was eliminated by SceI endonuclease digestion (from middle to right). C, After the PSCA-CAR construct was cloned into the parental plasmid DNA (pMC-Easy-PSCA-CAR), eight bacterial clones (lanes 1–8) were selected for restriction and sequencing analysis. Nearly all clones contained the PSCA-CAR construct (1,575 bp) except clone 2 (lane 2). D, Electrophoresis for the uncut, closed circular plasmid and minicircle DNA before (−) and after (+) L-arabinose induction. After L-arabinose induction, a lower band representing the mcDNA-PSCA-CAR was generated without the presence of the parental plasmid DNA band. M, molecular weight marker.
- Figure 2.
Efficient transfection of mcDNA-PSCA-CAR into human T cells and high expression of PSCA-CAR on the cell surface. A, Human T cells were transfected with pmaxGFP (electroporation control plasmid from the 4D-Nucleofector X kit), mock mcDNA control plasmid MN601A-1 (SBI), and mcDNA-PSCA-CAR, respectively. All three types of plasmid contain a GFP cassette which can be observed under a fluorescent microscope. B, Twenty-four hours after electroporation, transfection efficiency, as represented by the green fluorescence intensity, was evaluated by FACS. CD3-PE was used to label the T cells. Nonelectroporated normal T cells were used as a control. The statistical analysis of cells from three donors is shown on the right. C, Six hours after electroporation, the viability of T cells was assessed by FACS. In apoptotic or dead T cells (black dots), forward and side scattering were changed due to altered cell volume and increased intracellular granularity, as compared with those in healthy T cells (green dots). Statistics of three donors is shown on the right. D, Eight days after electroporation, the expression of PSCA-CAR was examined by FACS. Cells were incubated with protein L and then stained with SA-PE. (d′) The statistical analysis of D from three donors. E, Proliferation curves of normal T, mock T, and PSCA-CAR T cells. The statistical analysis was performed on cells from three donors. F, Eight days after electroporation, FACS results showed similar CD3, CD4, and CD8 expression profiles on normal T and transfected PSCA-CAR T cells. Statistics of three donors is shown on the right. For FACS results in B, D, and F, both the X and Y axes are log10 scales (100, 101, 102, 103). For the FACS analysis in C, both the X and Y axes, representing forward scattered (FSC-A) and side scattered (SSC-A) light respectively, are in Cartesian coordinates ranging from 0 to 250 (×1,000). Statistical analysis was performed by one-way ANOVA or Student t tests (ns, not significant; ****, P < 0.0001). Error bars represent SEM from triplicates.
- Figure 3.
PSCA-CAR T cells specifically targeted PSCA-positive cells and demonstrated strong cytotoxicity in vitro. A, FACS analysis of the surface expression of PSCA on RT4 and PC-3M human cancer cell lines. X axis is in the log10 scale; Y axis is in Cartesian coordinates ranging from 0 to 1250. B, Histograms showing the PSCA-specific cytotoxicity against RT4 or PC-3M cells in elevating effector-to-target cell ratios (2:1, 5:1, 10:1, and 20:1, respectively). Cells were obtained from three donors, tested 8 days after electroporation, and statistically analyzed. Statistical analysis was performed by nonparametric tests (ns, not significant; ***, P < 0.001; and ****, P < 0.0001). C, ELISA analysis showed PSCA-targeted secretion of IFNγ and IL2 against RT4 or PC-3M cells (effector-to-target cell ratio, 10:1). Cells were obtained from three donors, tested 8 days after electroporation, and statistically analyzed. Statistical analysis was performed using nonparametric tests or ANOVA (ns, not significant and ****, P < 0.0001).
- Figure 4.
In vivo systemic delivery of PSCA-CAR T cells suppressed tumor growth and demonstrated a persistent antitumor effect. A, PSCA expression levels in RT4- and PC-3M xenograft tumors. In each NOD/SCID immunodeficient mouse, 3 × 106 RT4 or PC-3M cells were injected. Fifteen days later, tumors were dissected and immunohistochemically analyzed using an anti-PSCA antibody. PSCA-positive staining showed a brownish color (magnification, 200×). B, Tumor size comparison. Tumor xenografts were excised 4 weeks after treatment with NS (no treatment control), normal T cells, or PSCA-CAR T cells. Three tumor samples were displayed for each treatment group. PSCA-CAR T cells were harvested 8 days after electroporation. C, Quantification of tumor volumes (mm3) in the three groups 4 weeks after the treatment. Nonparametric tests were used to compare the mean difference between each group at day 28 (****, P < 0.0001). D, FACS analysis revealed the presence of PSCA-CAR T cells in mouse peripheral blood 4 weeks after the treatment. Both the X and Y axes are log10 scales (100, 101, 102, 103). E, ELISA analysis of cytokine production (IFNγ and IL2) in mouse blood serum 4 weeks after the treatment. Statistical analysis was performed by nonparametric tests (****, P < 0.0001). F, IHC analysis of T-cell infiltration into the tumor xenografts. Four weeks after the treatment, tumor samples were excised from the mice, and divided for hematoxylin and eosin (H&E) and IHC analyses. For IHC analysis, tumor samples were stained with an anti-PSCA antibody or an anti-CD3 antibody; positive staining showed a brownish color (magnification, 200×).
Volume 19, Issue 1
